Biosafety of Recombinant
Viral Agents
Viral Vectors for Gene Therapy
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Introduction
Biosafety Principles
Biosafety Risk Categories
Biosafety Risk Assessment
Viral Vectors for Gene Therapy
• Classes of Viral Vectors
– Methods
– Applications
– Biosafety Considerations
Gene Therapy
• The transfer of genetic material to living
organisms:
– Correct a genetic defect
– Exert a phenotypic change
– Drug delivery
– Vaccines
• Viral and non-viral vectors
Biosafety – Basic Principles
• Primary containment
– protection of lab personnel and the
immediate laboratory environment
– laboratory practice and technique
• knowledge of infectious agents and risks
• strict adherence to standard microbiologic
techniques
• develop protocols and procedures to be
followed by all lab personnel working with
biologic hazards
Standard Operational Practices
1. Access to the laboratory is limited or restricted at the
discretion of the laboratory director when experiments or
work with cultures and specimens are in progress.
2. Persons wash their hands after they handle viable
materials,after removing gloves, and before leaving the
laboratory.
3. Eating, drinking, smoking, handling contact lenses,
applying cosmetics, and storing food for human use are
not permitted in the work areas. Persons who wear con
tact lenses in laboratories should also wear goggles or a
face shield .
Standard Operational Practices
4. Mouth pipetting is prohibited
5. Policies for the safe handling of sharps are instituted.
6. All procedures are performed carefully to minimize the
creation of splashes or aerosols.
7. Work surfaces are decontaminated at least once a day
and after any spill of viable material.
See Health Canada “Laboratory Biosafety Guidelines” for details
Standard Microbiological Practices
8. All cultures, stocks, and other regulated wastes are
decontaminated before disposal by an approved
decontamination method such as autoclaving.
9. A biohazard sign may be posted at the entrance to the
laboratory whenever infectious agents are present. The
sign may include the name of the agent(s) in use and the
name and phone number of the investigator.
Biosafety – Basic Principles
• Primary containment
– safety equipment
• bio-safety cabinets (class 2)
• centrifuge covers
• personal safety devices - gloves, goggles,
protective clothing, etc
Biosafety – Basic Principles
• Secondary containment
– protects personnel in the laboratory and
persons outside the laboratory
– facility design
Bio-Safety Levels
• Bio-Safety Level 1
– suitable for organisms not known to cause
disease in humans
– primary containment is standard
operational practices
– personal protection - eyeware, gloves,
labcoat, etc.
– other primary or secondary barriers are not
required
– sink for handwashing
Bio-Safety Levels
• Bio-Safety Level 2
– suitable for organisms known to cause
moderate disease in humans
– suitable for work involving human-derived
tissues and fluids
– hazard relates to accidental percutaneous
or mucous membrane exposure or
ingestion
Bio-Safety Levels
• Bio-Safety Level 2
– other primary or secondary barriers are not
required if the potential for aerosolization is
low
– bio-safety cabinets should be utilized if
there is a risk of aerosols or large
volumes/concentrations of infectious agent
are used
– Restricted access to infectious agents
(locked up)
Bio-Safety Levels
• Bio-Safety Level 2
– primary containment is standard
operational practices
– personal protection - eyeware, gloves,
labcoat, etc.
– waste decontamination
– sink for handwashing
Bio-Safety Levels
• Bio-Safety Level 3
– required for agents where respiratory
transmission is possible and cause serious
or potentially lethal disease in humans
– hazard to lab personnel relate to
autoinocluation and exposure to infectious
aerosols
– greater emphasis on primary and
secondary containment to protect lab
personnel
Bio-Safety Levels
• Bio-Safety Level 3
– all work must be performed in biosafety
cabinets or other enclosed equipment
– secondary containment includes restricted
lab access and special ventilation to
minimize release of infectious aerosols into
the surrounding environment
Bio-Safety Levels
• Bio-Safety Level 4
– suitable for rare and exotic agents that are
transmissible via aerosols, pose a high risk
of life-threatening disease, and for which
there is no known treatment or cure
– complete barrier between lab personnel
and the infectious agent is required
– special secondary containment facilities
Risk Groups
• Group 1
– Agents that are not associated with disease in adult humans
• Group 2
– Agents that are associated with human disease which is
rarely serious and for which there are preventative or
therapeutic measures often available
• Group 3
– Agents that are associated with serious or lethal disease in
humans for which preventive or therapeutic measures may
be available
• Group 4
– Agents that are associated with serious or lethal disease in
humans for which preventive or therapeutic measures are
not usually available
• Refer to Laboratory Biosafety Guidelines
manual from Health Canada
http://www.hc-sc.gc.ca/pphb-dgspsp/publicat/lbg-ldmbl-6/index.html
Biosafety – Basic Principles
• Risk assessment
– biology of wild-type virus
– prediction of biology of recombinant virus
vectors
• Principle investigator in conjunction with
the institution biosafety office is
responsible for assessing risk and
setting biosafety level
Biosafety - Risk Assessment
• Biology of wild-type virus
– Most vectors based on ubiquitous viruses, most of
which cause mild or no significant disease in
humans
– Route of transmission (potential for aerosol)
– Agent stability (capsid vs envelope)
– Animal viruses - some are controlled independently
by governmental regulations
Biosafety - Risk Assessment
• Recombinant vectors
– Human viruses adapted for gene therapy are
generally modified to reduce pathogenicity
• understand the origin and mechanism of attenuation
– Some recombinant vectors are still replication
competent
– Risk of generating replication-competent virus
• in vitro or in vivo through homologous sequences
• changes in selective pressure can produce viral mutants
– genetic variability
– Integrating vectors
Biosafety - Risk Assessment
• Recombinant vectors
– Genetic alteration of viruses
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tropism or host range
route of transmission
stability
pathogenicity or virulence
– Effect of the transgene
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toxins
oncogenes
suicide genes
cell cycle genes
genes that might increase the replicative capability of the
virus
• immune-modulating genes
Biosafety - Risk Assessment
• Recombinant vectors
– Consideration of the nature of the work
• Volume or concentration of vector
• Potential to produce aerosols
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Previous toxicity or animal studies
Availability of prophylaxis or therapy
Consideration of the likelihood of “rare” events
Animal transmission
Biosafety
Refer to “NIH Guidelines on
Recombinant DNA”
Viral Vectors
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Retrovirus
Adenovirus
Adeno-Associated Virus
Lentivirus
Other viruses
Retrovirus Vectors
• Moloney murine leukemia virus based
Retrovirus Vectors
Retrovirus Vectors
Retrovirus Vectors
Retrovirus Vectors
• Advantages:
– Broad tropism that can be altered via the
envelope protein
– Easy to generate (especially with VSV
pseudotype).
– Packaging cell lines available
– Accommodate up to 8 kb insert
– Integrates into host genome
– Non-immunogenic
Retrovirus Vectors
• Disadvantages:
– Unable to transduce non-dividing cells
– Integration into host is random, and thus
the potential for insertional mutagenesis
– LTR can affect activity of downstream
promoters
– Transgene expression unstable
Retrovirus Vectors
• Biosafety
– murine based vectors are BSL-1
• especially if produced in ecotropic cell lines
– risk of RCR depends on the packaging cell
line used
• viral genes separated into different transcription
units (env gene)
• amphotropic cell lines (lack endogenous
retroviral sequences)
– effects of transgene
Lentivirus Vectors
• Subgroup of the retrovirus family
– includes HIV
Lentivirus Vectors
Lentivirus Vectors
Lentivirus Vectors
• Advantages
– Infects dividing and non-dividing cells
• due to nuclear localization signals on the preintegration complex
– Risk of RC virus low
• absence of most HIV genes including env
– Integrates
– Silent LTR
– Non-immunogenic
Lentivirus Vectors
• Disadvantages
– RC virus production is not completely
eliminated
– Vectors can be rescued by wild type
infection. Creates the possibility of novel
infectious agents
– Insertional mutagenesis
Lentivirus Vectors - Biosafety
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Based on HIV (Group 3 agent)
Major risk is production of RCR
Effect of transgene
BSL-2+ or BSL-3 should be employed
Adenovirus Type 5 Genome
regulatory proteins
L1-4
L5
E1AE1B
E3
ITR
ITR
0 mu
100 mu
E2B
E2A
E4
Adenovirus Vectors
Adenovirus Vectors
Adenovirus Vectors
• Disadvantages
– Induce host inflammatory and immune
responses
– transient gene expression
– HD systems not completely devoid of
helper virus
Adenovirus Vectors
• Advantages:
– Can be grown in high titer
– Very efficient gene transfer
– Infects a variety of replicating and nonreplicating cells
– Epichromosomal, thus insertional
mutagenesis does not occur
– Helper-dependent systems can
accommodate over 30 kb of DNA
Adenovirus Vectors
Adenovirus Vectors
Adenovirus Vectors - Biosafety
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Group - 2 agent
Persists on surfaces for long periods
Transmitted via numerous pathways
Risk of aerosol
Effect of transgene
BSL-2 recommended
Adeno-Associated Virus
• Non-enveloped, single-stranded DNA
parvovirus
• Needs helper virus for replication
– adenovirus E2, E4
• Integrates into host genome to produce
latent infection
• No known disease in humans
Adeno-Associated Virus
Adeno-Associated Virus
Adeno-Associated Virus
• Advantages
– Broad tropism, infects dividing and nondividing cells
– low immunogenicity
– stable, long term gene transfer
– improved production methods has
eliminated helper virus contamination
– Neglible risk of RCR during production
Adeno-Associated Virus
• Disadvantages
– conversion to double stranded DNA rate
limiting step
– small, accomodates only 4.7 kb DNA
– does not integrate in the absence of rep
– can be rescued in vivo
– difficult to work with in vitro
Adeno-Associated Virus
• Biosafety
– AAV is a group 1 agent
– can be handled in BSL-1 conditions if not
produced with helper virus
– risk of aerosol
– prudent to use BSL-2
– effect of transgene
Other Virus-Based Vectors
• Herpesvirus
• Vaccinia Virus
• Alphavirus
– Semliki Forest and Sindbis
• Baculovirus
• Influenza
• Hybrids
The End